Newborn screening for X-linked adrenoleukodystrophy in New York State: diagnostic protocol, surveillance protocol and treatment guidelines.

PURPOSE: To describe a diagnostic protocol, surveillance and treatment guidelines, genetic counseling considerations and long-term follow-up data elements developed in preparation for X-linked adrenoleukodystrophy (X-ALD) newborn screening in New York State. METHODS: A group including the director from each regional NYS inherited metabolic disorder center, personnel from the NYS Newborn Screening Program, and others prepared a follow-up plan for X-ALD NBS. Over the months preceding the start of screening, a series of conference calls took place to develop and refine a complete newborn screening system from initial positive screen results to long-term follow-up. RESULTS: A diagnostic protocol was developed to determine for each newborn with a positive screen whether the final diagnosis is X-ALD, carrier of X-ALD, Zellweger spectrum disorder, acyl CoA oxidase deficiency or D-bifunctional protein deficiency. For asymptomatic males with X-ALD, surveillance protocols were developed for use at the time of diagnosis, during childhood and during adulthood. Considerations for timing of treatment of adrenal and cerebral disease were developed. CONCLUSION: Because New York was the first newborn screening laboratory to include X-ALD on its panel, and symptoms may not develop for years, long-term follow-up is needed to evaluate the presented guidelines.

Attitudes toward the prenatal diagnosis of cystic fibrosis: factors in decision making among affected families.

The objective of this study was to explore psychosocial factors underlying decisions about use of prenatal diagnosis for cystic fibrosis (CF), among parents of affected children. Anonymous survey questionnaires, supplemented by voluntary interviews, were used at 12 CF centers in six New England states, for a consecutive sample of families of minor children visiting CF centers during a 4-mo period. In all, 227 (71%) of 318 families responded. We hypothesized that attitudes toward utilization would be affected by (a) intentions to have children, (b) knowledge, (c) perception of risk, (d) the health of the child with CF, (e) expectations about the child's future, (f) attitudes toward abortion, (g) insurance, (h) genetic counseling, and (i) sociodemographic factors (including attendance at religious services). Of the 227 couples who responded, 69% were surgically sterile, over 45 years of age, widowed, or divorced, and 31% were at risk. Of 70 at-risk couples, 44% intended to have more children; of these, 77% had had or were considering CF prenatal diagnosis. Most families knew CF could be diagnosed prenatally; 20% would terminate for CF. Among intended prenatal diagnosis users, 44% would carry a fetus with CF to term, 28% would abort, and 28% were undecided. Stepwise logistic regression showed three variables significantly related to intentions to use prenatal diagnosis: (1) respondent's willingness to abort for CF (P less than .02, odds ratio 3.36), (2) respondent's siblings' approval of abortion for CF (P less than .03, odds ratio 2.99), and (3) respondent listed no accomplishments for the child with CF (P less than .09, odds ratio 3.01). The majority of affected families reject selective abortion for CF; many will curtail childbearing rather than use prenatal diagnosis.

Attitudes toward abortion among parents of children with cystic fibrosis.

BACKGROUND: DNA prenatal diagnosis for cystic fibrosis (CF) has been available for parents of affected children since late 1985. METHODS: Using anonymous questionnaires, we surveyed 395 parents of children with CF at 12 New England CF centers with regard to 12 maternal or family situations and 11 fetal characteristics; 271 (68%) responded. RESULTS: The majority supported legal abortion in the first trimester for all 23 situations; 58% would abort for severe mental retardation (MR), 40% would abort for a genetic disorder leading to death before age five years, 41% for a child bedridden for life, 35% for moderate MR, 20% for CF and 17% for a severe incurable disorder starting at age 40 years. Few would abort for a disorder starting at age 60 years, for genetic susceptibility to alcoholism or for sex selection. Variables most strongly related to abortion for CF were attitudes of spouse, respondent's siblings, and CF doctor toward abortion for CF as well as infrequent attendance at religious services. CONCLUSIONS: Prenatal diagnosis may not reduce substantially the number of CF births to parents of CF children because most do not accept abortion for CF.

DNA analysis of cystic fibrosis genotypes in relatives with equivocal sweat test results.

Restriction fragment length polymorphism linkage analysis of cystic fibrosis (CF) is used primarily for pre-pregnancy family studies, prenatal diagnosis, and carrier testing among close relatives of an affected individual. We undertook to clarify the status of six individuals with borderline or elevated sweat chloride concentrations and a relative with CF by testing for haplotype sharing. Their families and physicians expressed concern about management of these generally asymptomatic individuals. We typed DNA from family members with pJ3.11, pXV2C, pKM19, pmetH, pmetD, and p7C22. Each family was fully informative, enabling us to track the CF region of chromosome 7. Our analysis identified five individuals from four families as CF heterozygotes. A sixth individual, whose maternal first cousin died from CF, has the same haplotype as six of his seven healthy siblings, and thus we predict that he is unaffected. These family studies are a novel application of an emerging genetic technology. DNA linkage analysis is useful for elucidation of the CF genotype in families where the clinical features are equivocal and management is an issue.

Fragile sites and high-resolution chromosome studies in multiple endocrine neoplasia type 2A.

Affected individuals from four kindreds with multiple endocrine neoplasia type 2A syndrome (MEN-2A), were studied for the possible existence of a specific fragile site that might be associated with the MEN-2A gene. The chromosomes were also examined with high-resolution banding with particular emphasis on those chromosomes (#1, 10, 20, and 22) that have been implicated by previous studies from several laboratories as being associated with this disease. There was no evidence for a unique fragile site or a unique high-resolution banding pattern in subjects with MEN-2A. These findings, in combination with all previous cytogenetic studies, indicate that it is unlikely that current techniques will be useful in developing a simple cytogenetic test for this disease.

Vitamin B12 responsive homocystinuria and megaloblastic anemia: heterogeneity in methylcobalamin deficiency.

A male infant with methyl-B12 deficiency (cblE) presented at age 6 weeks with lethargy, staring spells, and vomiting. He later became hypotonic and unresponsive to stimuli and required intubation and ventilation. He had homocystinuria and hypomethioninemia with megaloblastic anemia but normal serum folate and vitamin B12 concentrations. No methylmalonic aciduria was detected. Fibroblasts, cultured from the patient, were unable to grow in medium in which homocysteine replaced methionine and incorporated abnormally small amounts of [14C]-methyl-tetrahydrofolate but normal amounts of [14C]-propionate into protein. Methyl-B12 content of fibroblasts was low, while the adenosyl-B12 content was normal. Methionine synthase activity was decreased when the assay was performed under both optimal and suboptimal reducing conditions, suggesting heterogeneity in the cblE disease. The patient responded dramatically to hydroxocobalamin treatment. Homocystinuria disappeared after 10 days of therapy, and methionine was normalized after 3 weeks. Psychometric testing at age 15 months showed a developmental age of 9 months.

Prevention of fetal damage through dietary control of maternal hyperphenylalaninemia.

Maternal phenylketonuria is a new entity in obstetrics. If unrecognized and for this or other reasons untreated, it produces a substantial risk for fetal damage. Our knowledge of the pathophysiology of the fetal complications in maternal PKU is very limited, but the degree of maternal hyperphenylalaninemia seems to be important. The management differs from the other high-risk pregnancies in the need for a special diet beginning before conception. An effective program of dietary therapy designed in collaboration with a PKU clinic will reduce the likelihood of fetal damage and improve pregnancy outcome.

A comparison of fragile X expression in lymphocyte and lymphoblastoid cultures.

This study compares fragile X expression in peripheral blood lymphocyte cultures with expression in lymphoblastoid cell lines established from 23 individuals from families in which the fragile X is segregating. Most patients expressed the fragile X in lymphoblastoid cell lines treated with FUdR under optimal conditions at approximately the same frequency as in peripheral blood cultures from the same individual. No fragile X cells were seen in the lymphoblastoid cell lines from three phenotypically normal males who had transmitted the fragile X gene to offspring or in the lines from three phenotypically normal obligate-carrier females, all of whom were also negative in peripheral blood cultures. Two individuals, however, who expressed at high levels in peripheral blood lymphocytes expressed in lymphoblastoid cells only at low levels or not at all. We describe the considerations needed for the consistent demonstration of the fragile X in lymphoblastoid cell lines.

Decreased methylation rates of DNA in SV40-transformed human fibroblasts.

The rates of methylation of total cellular DNA and newly synthesized DNA were measured in four unrelated SV40-transformed human fibroblast lines and in four control parent fibroblast lines. Rates of methylation of total cellular DNA were decreased by a factor of 1.8-2.3 in the transformed cells relative to control cells. Methylation was largely (75%-87%) restricted to newly synthesized DNA in control and transformed fibroblasts, and methylation rates of newly synthesized DNA were diminished in transformed cells by 12- to 19-fold relative to control cells. Growth rates were similar in the normal and transformed cells. The cellular uptake of methionine and conversion to S-adenosylmethionine were similar in the normal and transformed cells, suggesting no major differences between the normal and transformed cells in the cellular transport of methionine, methionine S-adenosyltransferase activity, or the intracellular concentrations of methionine and S-adenosylmethionine. The diminished rates of DNA methylation that we have observed suggest a possible mechanism for altered gene expression and growth control in transformed cells.

Cytogenetic analysis of lymphoblastoid cell lines.

Cytogenetic abnormalities were discovered in more than half of 16 lymphoblastoid cell lines established from fragile X individuals and their relatives upon routine cytogenetic analysis of early passage cultures. Subsequently, a second series of lymphoblastoid lines was analyzed to determine if the aneuploidy was a characteristic of lymphoblastoid cell lines derived from fragile X families or a result of the use of cyclosporin A in the establishment of these lines. In the second series of 33 lymphoblastoid cultures, no aneuploid clones were found in the fragile X group, while two were detected in the control cultures, one in a line initiated with cyclosporin A and the other in a line established without cyclosporin A. We conclude that the abnormal clones in our preliminary series were not a characteristic of lines derived from fragile X families and probably not due to the use of cyclosporin A. However, the finding of chromosome abnormalities in a large proportion of lines during the first 3 mo of culture contrasts with previous reports of chromosome stability for the first 12-18 mo of cultivation and indicates that the chromosomes of lymphoblastoid lines should be monitored in any experiment in which a normal diploid complement is critical.

Antifolate-induced misincorporation of deoxyuridine monophosphate into DNA by cells from patients with the fragile X syndrome.

The fragile site at Xq27 is expressed in vitro under conditions that lead to decreased intracellular thymidine triphosphate concentration, a condition which has also been shown to promote the misincorporation into DNA of deoxyuridine monophosphate (dUMP) in place of thymidine. We tested for increased whole-cell misincorporation of dUMP as a possible molecular mechanism for the expression of the fragile X abnormality. Neither deoxyuridine triphosphatase nor uracil-DNA-glycosylase, the two enzymes that normally prevent the accumulation of dUMP in DNA, was deficient in fragile X syndrome cells. Misincorporation of dUMP occurred in comparably low levels in both normal and fragile X syndrome lymphoblasts. Although these results provide strong evidence against generalized misincorporation of dUMP in fragile X syndrome cells, a substantial real difference present at Xq27 might not be detected in these studies of whole cells containing the diploid chromosome complement.

Thymidylate metabolism in fragile X syndrome cells.

The observation that decreased thymidylate supply in vitro induces the expression of the Xq27 chromosome fragile site prompted us to examine cellular thymidylate metabolism. Using a sensitive enzyme assay for deoxyribonucleotide triphosphates, we found that the total cellular thymidine triphosphate pools in cell lines from fragile X patients and carriers do not differ from normal controls under either basal or folate-deficient conditions. This agrees with our earlier observation that the thymidylate synthase enzyme activities in crude cell extracts of five fragile X syndrome lymphoblast lines do not differ from those in normal controls under standard assay conditions. Although a difference in the amount of thymidine triphosphate available at the replication fork for DNA synthesis remains a possibility, our results indicate that a readily demonstrable defect in thymidylate metabolism is not present in fragile X syndrome cells.

Folate metabolism in cells from fragile X syndrome patients and carriers.

The in vitro folate sensitivity of the fragile site at Xq27 and the claims of a beneficial response of patients given folic acid prompted us to examine the folate metabolism in cells cultured from fragile X syndrome patients and carriers. Using Epstein-Barr virus we established permanent lymphoblastoid lines from 4 fragile X syndrome males and 3 carriers from 7 families. All these lines expressed the fragile site when 0.1 microM 5-fluorodeoxyuridine (FUdR) was added to the cultures 24 hr prior to harvest; thus, the lines seemed suitable for seeking an intrinsic defect. Fragile X syndrome patient and carrier lines and normal control cell lines did not differ in regard to folate requirement for growth, the ability to use homocysteine in place of methionine, the ability to utilize reduced folates as the sole folate source, or methotrexate sensitivity. These results suggest that no intrinsic defect in folate metabolism is present in fragile X syndrome cells.

Expression of the marker (X) (q28) in lymphoblastoid cell lines.

The marker(X)(q28) chromosome associated with one type of X-linked mental retardation has been demonstrated in lymphoblastoid cell lines established from affected individuals. The mar(x) can reliably and repeatedly be seen by the addition of FUdR to the cultures for 24 hrs prior to harvest. This simple technique provides an excellent in vitro experimental test system for investigation of the mar(X).

Decreased purine synthesis during amino acid starvation of human lymphoblasts.

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.

Folate polyglutamate and monoglutamate accumulation in normal and SV40-transformed human fibroblasts.

Folate polyglutamate and monoglutamate accumulation was measured in normal diploid and SV40-transformed human fibroblasts by Sephadex G-10 gel filtration chromatography. The cells were first depleted of folates and then provided with limiting amounts of [3H]-folic acid in order that the cells would accumulate only forms of folate necessary for proliferation. Both the normal and the transformed cells accumulated monoglutamate and polyglutamate forms, but by 72 hours of labeling the transformed cells contained 3-10 times more polyglutamate than the normal cells. The growth rates for the normal and transformed cells were similar at this limiting folic acid concentration. Thus, if folate polyglutamates are more important for the proliferation of SV40-transformed cells than the normal cells, then inhibition of polyglutamate formation may be an important potential target for chemotherapy.

Decreased rates of methionine synthesis by methylene tetrahydrofolate reductase-deficient fibroblasts and lymphoblasts.

Methionine synthesis from homocysteine was measured in intact human fibroblasts and lymphoblasts using a [14C]formate label. Seven fibroblast lines and two lymphoblast lines derived from patients with 5,10-methylene tetrahydrofolate reductase deficiency had rates of methionine synthesis that were from 4 to 43% of normal. When the patients were divided by clinical status into mildly (two patients), moderately (two patients), and severely (three patients) affected, methionine biosynthesis expressed as a percent of control values was 43 and 33%, 11 and 10%, and 7, 6, and 4%, respectively, in fibroblasts. Similar data for the two lymphoblast lines were 36 and 26% for a mildly and moderately affected patient, respectively. These data are to be contrasted with the measurement of residual enzyme activity in cell extracts which agrees less precisely with the clinical status of the patients. In the presence of normal methionine synthetase activity, the rate of synthesis of methionine from homocysteine is a function of the activity of the enzyme 5,10-methylene tetrahydrofolate reductase, and measurement of the methionine biosynthetic capacity of cells deficient in this enzyme accurately reflects the clinical status of the patient from whom the cells were derived.